Development of a temporally controlled adjuvant system to adenovirus gene-directed enzyme prodrug therapy (GDEPT) represents a new platform in anticancer therapy. Matrix-mediated delivery of adenovirus effectively manages tumor growth while limiting therapeutic drawbacks, including transient gene expression, non-target viral dissemination, and immune activation. Adenoviruses readily transfect many cell types but increased transfection efficiency is achieved when co-delivered with a polycation. Temporal, controlled delivery of a polycation alongside adenovirus will decrease the effect of the transient expression and prolong therapeutic efficacy. Silk-elastinlike protein polymers (SELPs) form hydrogel matrices for release of adenoviruses within a local tumor environment. Although local expression is increased compared to viral injection alone, this system suffers from decreasing levels of transgene expression from week to week. Temporal release of the polycation protamine will increase expression levels at later time points. The objective of the proposal is to develop a distinct system for temporal and environmental release of an adjuvant to establish a platform delivery system for improved adenovirus GDEPT for anticancer therapies. The utility of such a gene delivery matrix promises increased therapeutic outcomes and reduction of toxicity. This proposal introduces matrix-metalloproteinase (MMP) responsive amino acid linkers to tether protamine to SELP hydrogel backbone and to release this adjuvant in a temporal manner. By introducing protamine to be released in a MMP-responsive manner, local adenovirus transfection potentially will increase to lead to improved efficacy. The following Specific Aims will lead to the development of this system of adenovirus delivery. The first aim will be to synthesize, purify, and characterize linear MMP-degradable silk-elastinlike protein polymers containing functionalized protamine analogs. The second aim will be to evaluate the gelation properties, release profile of protamine and adenoviruses, and transfection efficiency of protamine-functionalized polymers in vitro. The third aim will establish the extent of improvement of the temporal expression profile of a marker gene from adenovirus using functionalized SELP hydrogels in vivo through intratumoral injections. The key novelty of the proposed research is the inclusion of temporally released protamine as an adjuvant to matrix mediated adenoviral GDEPT. The important innovation in the proposed fellowship is the establishment of a temporally released adjuvant to viral GDEPT. The development of this proposal and training to be carried out by the PI will establish a new approach in enhancing local adenoviral delivery and increasing anticancer therapeutic efficacy.